Genetical genomics of Puccinia graminis TTKS response: A systems biology approach to rapid development of durable resistance for barley and wheat Roger Wise USDA-ARS / Iowa State University http://wiselab.org/ http://plexdb.org/ http://barleygenome.org/
Gene Expression and QTLs The treatment of transcript accumulation as a Quantitative Trait is called eQTL mapping. Loci that regulate expression of genes or networks can be: -cis-acting QTL -trans-acting QTL
Requirements • Experimental population that harbors genetic variation for resistance to the pathogen • [QSM DH (Q21861 x SM89010)] • Global gene expression profiling platform • (Affymetrix Barley1 GeneChip) • High-throughput genotyping platform • (QSM SFPs detected by Barley1 GeneChip) • Database (BarleyBase/PLEXdb)
Scoring of TTKS on QSM DH mapping population 0; 12 21 3-210; 3+ Brian Steffenson
Barley1 GeneChip22,840 Probe Sets (22,792 + controls) Close et al. 2004, Pl Physiology 134: 960-968
Single feature Polymorphisms (Steptoe and Morex) 22,000 Genes X 11 Oligos X 25nt = 6,325,000 bases [ >4,000 SFPs ] Steptoe Raw Data: Morex Robbie Waugh
Expression Level Polymorphism Contig3660_at Matt Moscou
Experimental Design Dew Chamber QSM DH population (79 lines) Diversity Set (12 lines) Red Trays- Inoculated Blue Trays-Mock-inoculated RNA from each cone of seedlings is hybridized to its own Barley1 GeneChip. Each flat contains up to 98 cones of (6) seedlings randomly arranged among reps and inoc & mock-inoc (Harvested 24 hours after inoculation). [79 DH lines + parents] x 2 inoc. treatments x 2 reps = 340 randomized samples 12 diversity lines x 2 inoc. treatments x 6 reps [= 144 randomized samples]
Data Analysis • Identify subset of genes that show differential expression between TTKS-inoculated and mock-inoculated leaves. • Map the eQTL that regulate the expression of these genes. • Identify regulators by hierarchical analyses. • A locus that regulates one or more significant disease resistance networks is an obvious target for immediate use in breeding.
Expectations • 4,000-8,000 SFP gene markers positioned on the barley population segregating for TTKS resistance; enables QTL mapping of TTKS resistance. • Marker-delineated positions of the eQTL that regulate networks of disease response genes; breeders can incorporate into their germplasm. • Elucidation of gene expression networks responsive to TTKS infection; provides biochemical targets for further analysis. • Genes that possess transcriptional activation elements that allow them to respond more aggressively to TTKS infection; provides key regulators for disease response.
What do we do with all that data? (65 Mb/chip = > 30 GigaBytes)
Knowledge For Tomorrow’s Solutions USDA-ARS/ISU Rico Caldo Matt Moscou Greg Fuerst Nick Lauter Jose Rodriguez Yan Meng Pingsha Hu Liu Xi Dennis Halterman Karin Werner Brent Kronmiller BarleyBase/PLEXdb Julie Dickerson Ethy Cannon Sudhansu Dash Lu Hong Lishuang Shen Dan Nettleton USDA-ARS/UMN Yue Jin Sam Gale Les Szabo Univ. of Minnesota Brian Steffenson Stephanie Dahl